The cholesterol-dependent cytolysins (CDCs) are a large group of pore-forming toxins that contribute to the pathogenesis of many Gram-positive pathogenic bacteria. Our studies of the CDC pore-forming mechanism have led to a better understanding of their role in pathogenesis and have revealed new paradigms for cellular recognition and assembly of a membrane pore. These studies enabled us to rationally design a CDC-based component vaccine for Streptococcus pneumoniae, which is currently being used by the Gates Foundation in their vaccine effort for S. pneumoniae. Also, our discovery of a CD59 binding CDC has allowed others to develop a possible therapeutic that specifically targets HIV. In the current renewal we will continue our studies into the CDC mechanism to understand the molecular events that trigger pore formation. Understanding how the CDCs initiate pore formation will help us design therapies or improved vaccines that specifically target highly sensitive structural components necessary for their function. We will also continue our studies into the membrane recognition of cholesterol by the CDCs. Our studies have revealed a remarkable aspect of the CDCs' interaction with cholesterol: some CDCs can bind to an expanded population of membrane cholesterol of which only a fraction supports pore formation. It appears that the ability to bind a significant fraction of monomers to cholesterol at sites that do not support pore formation may be important to specific CDC functions. It was recently shown that the CDC of Streptococcus pyogenes, streptolysin O (SLO), transports a specific protein into eukaryotic cells by a pore-independent mechanism. These studies suggest that pore- independent effects of the CDCs may be a significant, but unexplored, mechanism by which the CDCs contribute to pathogenesis. Understanding how CDCs can bind to membrane cholesterol without triggering pore formation will reveal new paradigms for how CDCs function at the molecular level and how they contribute to pathogenesis. Finally, we will leverage our deep understanding of the CDC mechanism to initiate studies into the pore forming mechanism of the membrane attack complex/perforin (MACPF) family of proteins. The MACPF proteins are widespread and contribute to mammalian immune defense and disease causing prokaryotic and eukaryotic pathogens. Recent structural studies of MACPF proteins and our work on the CDCs has prompted others to speculate that the MACPF pore forming mechanism exhibits features of the CDC pore forming mechanism, possibly resulting from a common origin of the two protein families. We will leverage our expertise with the CDCs to initiate studies into the molecular mechanism of the MACPF proteins to test this hypothesis. We expect the study of the MACPF pore forming mechanism will form the basis of a significant effort to investigate other MACPF proteins that play important roles in immune defense (complement membrane attack complex), development (astrotactins), diseases caused by eukaryotic pathogens such as Toxoplasma and malaria, and tumor destruction (perforin).